Estrogen receptor (ER) expression has important implications for the prognosis and treatment of breast cancer. Patients with tumors that express ER have improved mortality, a longer disease-free interval, and are more likely to respond to hormonal therapy than patients with rumors that lack ER expression. In both breast carcinoma cell lines and primary breast tumors, ER expression is largely controlled by transcription of the ER gene. Transcription of the human ER gene is regulated by activity from the main ER promoter, P1, and at least two alternate ER promoters, both of which are active in breast carcinoma cell lines and primary breast cancers. Our previous work had identified the ERF-1 transcription factor involved in regulation of the main ER gene promoter in breast carcinomas. We have recently cloned ERF-1 and demonstrated that it is a member of the AP2 transcription factor family. The goal of this grant proposal is to determine the molecular basis for the improved prognosis associated with the hormone-responsive breast cancer phenotype. There are three aims of this grant proposal. Fist, we will identify the regulatory elements of alternate ER promoters. This will be accomplished by identifying the cap sites of the alternate ER transcripts, cloning genomic regions near the cap sites upstream of a receptor gene to identify cis-regulatory elements, and using gel shift and footprinting to identify factors regulating expression of the alternate ER transcripts. Second, we will demonstrate that ERF-1 regulates transcription from the main and alternate ER promoters. This aim will be accomplished by co-transfecting the cloned ER promoters with the ERF-1 gene and by studying the effect of ERF-1 expression on the native ER gene in breast cancer lines. Third, we will characterize the expression of novel ER-regulated genes and determine their function in hormone-responsive tumors. This will be accomplished by cloning and sequencing cDNAs of novel ER-regulated genes and characterizing primary breast tumors for expression of these genes.